Tissue, Cell and Callus Culture of Pterocladia and Porphyra Seaweeds

Abstract

Two species of Pterocladia used for agar production in New Zealand, P. capillacea and P. lucida, were studied with respect to tissue, cell and callus culture in order to obtain information on the vegetative propagation of these two species and requirements for regeneration of plants. Two methods for establishing unialgal culture from wild Pterocladia and a sterilizing method for obtaining axenic material are described in detail. The regeneration ability of 1-mm Pterocladia segments was studied, and also the effects of the synthetic hormones NAA and kinetin on this regeneration were examined. The results showed that 1-mm segments of Pterocladia were able to regenerate into a quite normal plant except for the lack of a holdfast, and the hormones had little effect on the regeneration pattern. A friable callus was obtained from P. capillacea segments cultured in PES medium with 3μml-1 NAA added. This is the first time that callus has been obtained from this species. The callus failed to regenerate a new plant in culture. However, obvious effects of hormones on callus cell shape and callus cell division and development were noted and are described in detail. Single cells were isolated from both species of Pterocladia by using an enzyme solution extracted from the N.Z. sea snail Melagraphia aethiops. The procedure for preparing the enzyme solution is also presented. The fluorescent staining method was used to examine the state of the cell wall of Pterocladia tissue, cells and callus. Studies on the edible alga, Porphyra columbina, involved developing techniques for cryopreservation of thalli and production of single cells for micropropagation. In order to determine a successful cryopreservation method, a series of pretreatment methods was tested. Living Porphyra thalli were obtained from frozen Porphyra which had been exposed in air for 24, 48 or 72 hours, or immersed in sea water with 0.4 M NaCl added, before being frozen for 13 months at -16°C. Viability of recultured frozen Porphyra thalli was determined by observing 1) if any bubbles were released from thalli indicating active photosynthesis; 2) if plasmolysis occurred when pieces of thallus were put in a medium of lower osmotic potential 3) if any carporspores were released from the thalli. Living single cells were isolated from both fresh and frozen Porphyra thalli by using enzyme solution described in the Pterocladia section.